Medicinal Research Reviews - 2022 - Li - Toward The Next Generation of Vascularized Human Neural Organoids

Received: 20 October 2021 | Revised: 22 February 2022 | Accepted: 9 August 2022
DOI: 10.1002/med.21922
REVIEW ARTICLE
Toward the next generation of vascularized

human neural organoids
Minghui Li1,2 | Lixiong Gao3 | Ling Zhao4 | Ting Zou1,2 |

1,2
Haiwei Xu
1
Southwest Hospital/Southwest Eye
Hospital, Third Military Medical University Abstract
(Army Medical University), Chongqing, China
Thanks to progress in the development of three‐
2
Key Lab of Visual Damage and Regeneration
& Restoration of Chongqing, Chongqing,
dimensional (3D) culture technologies, human central
China nervous system (CNS) development and diseases have
3
Department of Ophthalmology, Third been gradually deciphered by using organoids derived from
Medical Center of PLA General Hospital,
Beijing, China human embryonic stem cells (hESCs) or human induced
4
State Key Laboratory of Ophthalmology, pluripotent stem cells (hiPSCs). Selforganized neural orga-
Zhongshan Ophthalmic Center, Sun Yat‐sen noids (NOs) have been used to mimic morphogenesis and
University, Guangzhou, China
functions of specific organs in vitro. Many NOs have been
Correspondence reproduced in vitro, such as those mimicking the human
Haiwei Xu, Southwest Hospital/Southwest
Eye Hospital, Third Military Medical brain, retina, and spinal cord. However, NOs fail to
University (Army Medical University), capitulate to the maturation and complexity of in
Chongqing, China; Key Lab of Visual Damage
and Regeneration & Restoration of vivo neural tissues. The persistent issues with current NO
Chongqing, Chongqing 400038, China. cultivation protocols are inadequate oxygen supply and
Email: haiweixu2001@163.com
nutrient diffusion due to the absence of vascular networks.
Funding information In vivo, the developing CNS is interpenetrated by vascula-
National Natural Science Foundation of
ture that not only supplies oxygen and nutrients but also
China, Grant/Award Numbers: 31930068,
81873688; National Key Research and provides a structural template for neuronal growth.
Development Program of China,
To address these deficiencies, recent studies have begun
Grant/Award Number: 2018YFA0107302
to couple NO culture with bioengineering techniques and
methodologies, including genetic engineering, coculture,
multidifferentiation, microfluidics and 3D bioprinting, and
transplantation, which might promote NO maturation and
create more functional NOs. These cutting‐edge methods
could generate an ever more reliable NO model in vitro for
deciphering the codes of human CNS development, disease
progression, and translational application. In this review, we
Med Res Rev. 2023;43:31–54. wileyonlinelibrary.com/journal/med © 2022 Wiley Periodicals LLC. | 31

10981128, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/med.21922 by Shenzhen University, Wiley Online Library on [24/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
32 | LI ET AL.
will summarize recent technological advances in culture

strategies to generate vascularized NOs (vNOs), with a
special focus on cerebral‐ and retinal‐organoid models.
KEYWORDS
human neural organoid, angiogenesis, organoid vascularization,
cortical organoid, retinal organoid
1 | INTRODUCTION
Over the past few decades, the advent of organoid culture systems has revolutionized in vitro studies of human
embryonic tissue development, including the brain, liver, lungs, kidneys, and intestines. Unlike other organ
systems of the human body, the unique features of the central nervous system (CNS) distinguish humans from
other animal species. Studies of neurogenesis, neural processes, and neurological disorders have always
fascinated scientists; however, studies are limited by ethical and practical issues, especially concerning
harvesting developing brain tissues from fetuses. Human pluripotent stem cells (hPSCs), including human
embryonic stem cells (hESCs) and human‐induced pluripotent stem cells (hiPSCs), represent the capability to
differentiate into any cell type in vitro, making them as invaluable tools for studying human CNS development
and the pathobiology of its disorders. Under specific and favorable culture conditions, stem cells can
selforganize in vitro and undergo an in vivo‐like morphogenesis to generate three‐dimensional (3D) organ‐like
tissues (organoids) that contain multiple specific cell types. Neural organoids (NOs) recapitulate the key
features of neurogenesis, therefore providing a new platform to study CNS development and disorders.1–3 NOs
can resemble the embryonic or fetal human brain with high spatiotemporal complexity, not only exhibiting
dynamical structural and transcriptional changes but also reflecting the development of structured network
activity.4–9
hPSC‐derived NOs have been used as an autologous source for cell or tissue transplantation in regenerative
medicine. Although carrying out NO‐based treatment remains controversial, NOs indeed contain various
types of neurons such as neural progenitor cells (NPCs), which hold great promise for cell replacement
therapy in human neurological diseases. Organoid‐derived NPCs have selfrenewal capacity and differentiate
into various cell types, and promote the selfrenewal capacity and differentiation potential of host neural stem
cells in regenerative neurogenesis. Our previous study has demonstrated that retinal organoid (RO)‐derived
C‐Kit + /stage‐specific embryo antigen 4− negative (SSEA4− ) human retinal progenitor cells (RPCs) provided a
protective retinal microenvironment after subretinal transplantation into rodent models with retinal
degeneration. 10 Nevertheless, the mechanism regulating the integration of engrafted neurons into the
injured neural circuit is still debatable. The use of NOs in regenerative medicine could also be combined with
genetic editing, such as clustered regularly interspaced short palindromic repeats (CRISPR), to correct native
genetic disorders.
The pathogenesis and mechanisms of human neurological diseases and disorders such as Alzheimer's disease
(AD), autism spectrum disorders (ASDs), and Parkinson's disease (PD), are complex, which makes early diagnosis and
effective treatment of these diseases challenging. Recently, organoids have been used as ideal models for studying
the pathophysiology of human CNS diseases. In particular, patient‐derived hiPSCs were utilized to model
neurodevelopmental conditions (Microcephaly and ASD), psychiatric diseases, and neurodegenerative diseases
(AD and PD).11–15 Therefore, NOs open up a new door to neuroprotective‐drug discovery in CNS disorders by
establishing disease models. For instance, brain organoids infected by the Zika virus (ZIKV) not only
showed features of congenital Zika syndrome include decrease of neuronal cell‐layer volume, disruption of
LI ET AL. | 33
apical‐surface adherent junctions, and dilation of the ventricular lumen, but also hold great potential for anti‐
ZIKV drug testing.11,16–18 Recently, NOs have been applied in exploring the underlying mechanisms of
neurological complications caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2)
infection.19,20 In addition, NOs have potential applications in testing the efficacy of drugs and the toxicity of
environmental compounds.1,21–23
NOs have emerged as powerful research tools to better uncover human brain evolution, but they currently
possess several limitations, including the absence of a circulatory system and an immune microenvironment
(especially microglia), as well as poor reproducibility. Therefore, current NOs cannot fully mimic realistic
neurodevelopmental conditions that are important to their growth and maturation. To tackle these challenges,
many strategies have been invented to improve the functionality and maturation of the resulting organoids. For
instance, bioreactors have been extensively used to improve culture conditions by promoting the diffusion of
oxygen and nutrients via constant culture spinning, and to promote neuronal survival and gradual maturation.24,25
Cerebral organoids can be maintained for over 1 year in long‐term culture in such bioreactors, and as a result, their
structures resemble more closely to the developing human brain.26 Vascularized and functional organoids have also
been demonstrated by transplanting them into host animals, where native vasculature integrates into the
neurovascular networks of the ectopic implant.27,28 Under these conditions, organoid grafts progressively
differentiated and mature, leading to functional neuronal networks that are synaptically interconnected with host
neuronal circuits.27,28 Furthermore, bioengineering organ‐on‐a‐chip devices combined with 3D bioprinting and
mathematical modeling help generate more‐physiologically‐relevant human organoids and bridge the gap between
organoids and native organs. In this review, we discuss the technological advances in generating vascularized NOs
(vNOs), including cortical organoids (COs) and ROs, that mimic native tissue. We also present obstacles to be
overcome and future directions in the field.
2 | D E V E L O P M E N T OF NE UR A L O R G A N O I D S
The early embryonic CNS begins as a simple neural plate that folds to form a neural groove and neural tube in which
stem cells generate neurons and glia.29,30 In 2001, Zhang et al. demonstrated that hESC‐derived human neural
rosettes have apicobasal polarity and epithelial characteristics similar to those of the embryonic neural tube
(Figure 1).34 Neural rosettes developed in vitro can lead to the sequential generation of intermediate progenitor
types and even rough progenitor zones that were similar to the ventricular and subventricular zones (VZ/SVZ) in
vivo.35 Ying et al. described a protocol to induce neural differentiation of ESCs in the complete absence of
exogenous growth factors or other inductive signals in the medium.36 Based on this protocol, Watanabe et al.
showed that the embryoid body (EB) cultured with growth factor‐free chemically defined medium, could generate
specialized neural subtypes.37 Sasai et al. further developed this method into the serum‐free floating culture of EB‐
like aggregates with quick reaggregation (SFEBq).38 In this culture system, both mouse and human embryonic stem
cells (mESCs/hESCs) could spontaneously form polarized cortical tissue in vitro. In 2009, Chambers et al. devised a
method for high‐efficiency neural rosette induction by applying dual inhibition of SMAD signaling (dual‐Smad)
(Figure 1).39 Subsequently, the SFEBq approach has been successfully applied to the further development of
individual brain regions, including the retina, adenohypophysis, cerebellum, forebrain, hippocampus, midbrain, and
hypothalamus.26,30,40–46 Most recent studies have turned to region‐specific organoids and moved away from
whole‐brain organoids because of the unguided protocols for producing the latter, leading to considerable
variability.3,45,47,48 Interestingly, Gabriel et al. established novel human brain organoids that assembled functionally
integrated bilaterally symmetric optic vesicles, which developed progressively as visible structures in the following
culture, including primitive corneal epithelial and lens‐like cells, retinal pigment epithelium (RPE), RPCs, axon‐like
projections, and electrically active neuronal networks.33,49
34 | LI ET AL.
F I G U R E 1 Timeline of neural organoid development over the past decade. Generation of the neural rosette in
2001; generation of optical cup organoids; generation of cerebral organoids in 2013; Immunofluorescence staining
of whole‐mount vascularized organoid on Day 42, reproduced with permission.31 Generation of neural‐vascular
organoids by coculturing with mesodermal progenitor cells in 2020. Generation of mature human ROs in 2020,
reproduced with permission.5 Assembly of 3D cultures derived from hiPSCs resembling the retina and brain,
reproduced with permission.32 Generation of optic vesicle‐containing brain organoids, reproduced with
permission.33 GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer;
OPL, outer plexiform layer. [Color figure can be viewed at wileyonlinelibrary.com]
2.1 | Generation and characterization of cortical organoids
The human cerebral cortex is the most uniquely human organ: its threefold expansion is one of the most
conspicuous features distinguishing humans from animal models.3,50,51 The developing human neocortex exists the
expansion of progenitor cells into the outer subventricular zone (oSVZ), which is a highly complex and dynamic
process involved in neuronal proliferation, differentiation, and migration.52,53 The oSVZ also contributes to the
evolutionary increase in human‐cortex size and complexity.54,55 Moreover, the human oSVZ radial glial‐cell
phenotype differs even from those of our closest primate relatives, suggesting the evolutionary expansion of the
human neocortex.51,52,56 Interestingly, Sasai's SFEBq protocol enabled the formation of a selforganized 3D cortical
structure containing four distinct zones: ventricular, early and late CP, and Cajal Retzius cell zones in the apico‐basal
direction.38 These generated neurons recapitulate the time course of corticogenesis, and the cortical structures are
spatially organized into VZ‐, SVZ‐, and CP‐like regions presenting initial cortical layering and various aspects of the
early developing human neocortex.57 Therefore, human COs are potentially useful to identify the cell types and
structural features of the human cerebral cortex, and the effects of pathogenic mutations that are difficult to model
in animals.58
In 2013, Lancaster et al. reported that Matrigel embedding supports the selforganization of neuroepithelium
and that formed organoids recapitulate the gene expression and morphology of the early developing human
LI ET AL. | 35
neocortex.46 In a recent study, Yoon's group generated cortical spheroids that were highly reliable and consistent
by single‐cell RNA sequencing (scRNA‐seq).59 Moreover, these cortical spheroids were functional and could be
maintained for long periods (over 100 days) in vitro to capture human corticogenesis. Given cell interaction and
migration in the developing forebrain, fused organoids of the dorsal and ventral forebrain have been denoted
“assembloid,” each with two distinct but interfacing domains (Figure 1).60–63 Cortico‐motor assembloids (three‐part
assembloids resembling the human cerebral cortex, hindbrain/cervical spinal cord, and skeletal muscle) enable the
formation of functional neural circuits that can be readily manipulated in the model cortical control of muscle
contraction in vitro for up to 10 weeks postfusion.64 However, current organoids are limited by the free diffusion of
oxygen and nutrients.65 Ming and colleagues overcome the diffusion limit by slicing COs into smaller pieces to
increase nutritional support and oxygen exchange under constant agitation, leading to the formation of well‐
separated upper and deep cortical layers.21 This method resulted in sustained neurogenesis and an expanded CP.
Given the adequate delivery of oxygen and nutrition, accumulating evidence suggests that the vascularization of
COs is of great importance.
2.2 | Generation and characterization of retinal organoids
The retina, which is formed from brain tissue during development, is considered to be part of the CNS.
Groundbreaking discoveries in stem cell research have greatly facilitated in vitro differentiation of RPCs and their
derivatives from ESCs in two‐dimensional (2D) adherent cultures.66,67 Subsequently, research following Sasai's
protocol has proven the potential of mESCs and hESCs to form a 3D optic cup structure.40,41 Selforganized ROs
contain all major retinal cell types and exhibit stratification of neuronal layers. Two major approaches have merged
to generate human ROs, including 3D, and a combination of 2D and 3D (for an excellent review, see Kruczek and
Swaroop68). Compared with 3D‐only culture, the combined 2D/3D approach improves the yield and efficiency of
RO generation and achieves higher organization and maturity levels in late‐stage organoids.68–70 In the wake of
these seminal reports, several other studies reported the derivation of ROs from iPSCs under different culture
conditions. Notably, both ESCs and iPSCs could be successfully and comparably used to generate human ROs.71,72
In 2014, hiPSCs were successfully inducted by Zhong et al. to generate fully laminated ROs containing mature
photoreceptor outer segments that demonstrated a certain electrical response on light stimulus.69 Mellough et al.
showed that exogenous insulin‐like growth factor 1 (IGF‐1) treatment increased the formation of correctly
laminated retinal tissue composed of multiple retinal phenotypes, as well as accelerated photoreceptor
maturation.73 In another report, the addition of COCO, a multifunctional antagonist of the Wnt, transforming
growth factor‐beta (TGF‐β), and bone morphogenetic protein (BMP) pathways increased the differentiation
efficiency of photoreceptor precursors and cones in hiPSC‐derived ROs.74 Decellularized extracellular matrix (ECM)
from retinal tissue could guide retinal cell adhesion, migration, organization, and enhance the maturation of ROs.75
In 2020, Cowan et al. generated ROs with three nuclear and two synaptic layers, showing that photoreceptors
could respond to and transmit visual information synaptically in second‐ or third‐order retinal cells.5 Recently, ROs
were generated and organized with brain organoids into an assembloid that models the projections of the visual
system (Figure 1).32 In this model, retinal ganglion cells (RGCs) could extend axons deep into brain organoids under
environmental cues, providing a novel platform for studying the development of human RGCs. Thus, ROs have been
used to investigate the physiology of RGCs in retinal degenerative diseases, such as glaucoma and retinitis
pigmentosa.76,77
hiPSC‐derived ROs have been applied for retinal disease modeling and drug discovery. The reprogramming of
patient somatic cells into iPSCs has opened new doors for investigating the pathogenic mechanisms underlying
retinopathies, including retinitis pigmentosa and age‐related macular degeneration.77,78 Defects or gene mutations
in early development are modeled on ocular maldevelopments, such as the visual system homeobox 2 (VSX2)
mutation (p.Arg200Gln) associated with microphthalmia.79,80 It is exactly based on the notion that ROs are valuable
36 | LI ET AL.
tools for screening small‐molecules in the treatment of various retinal diseases.81–83 In the field of translational
medicine, ROs have been widely used in cell replacement therapy approaches.68,84–90 The grafted organoid‐derived
cells or sheets can mature and form synaptic connections between the graft and host following transplantation, but
the functionality of the transplant itself is difficult to attain.86,89–91 More convincing and direct evidence is needed
to demonstrate the efficacy of therapeutic strategies in human ROs.92 Moreover, ROs are also promising tools to
evaluate the potential adverse effects of toxic compounds on human retinal development.22,93
3 | CURRENT CHALLENGES OF HUMAN NEURAL

O R G A N O I D D E V E L O PM E N T
The efficacy of CNS organoids as models is constantly improving with continued optimization of protocols.
However, there remain several limitations, including the absence of certain cell types in vivo, high levels of organoid
heterogeneity, and immaturity. The first issue with current COs and ROs is the absence or late emergence of
specific cell types that differ from those found in vivo. Neurons, microglia, and ECs are essential for normal CNS
neurogenesis, development, and maintenance of functional homeostasis. Unlike neurons, which are derived from
ectoderms, microglia (formed in the yolk sack) must migrate into the CNS before the blood‐brain barrier (BBB)
forms.94–96 Microglia, the primary immune cells of the CNS, not only maintain the homeostasis of the immune
system but also provide diverse surface membrane receptors for receiving and processing myriads of signals in the
microenvironment.96–98 Recently, several studies have successfully demonstrated the differentiation of hiPSCs into
microglia‐like cells, which could be integrated into brain organoids.99–105 In hiPSC‐derived cerebral organoids,
mesodermal progenitors can develop into microglia‐like cells that exhibit a microglia‐specific molecular signature
and mediate phagocytosis.106 These studies opened new doors for researchers studying the possible roles of
microglia in CNS development and disease pathology.102
Another challenge is that current organoids are characterized by high variability and heterogeneity, reflected in
their different sizes, shapes, and cytoarchitectures.30,107,108 Several reasons can be considered. First, brain
organoids derived from the cellular selforganization process can lead to the stochastic formation of specific regions
differing in construction and morphological variability among various tissue areas.30 Second, brain organoids display
opposing ranges of homogeneity and reproducibility. Third, the intrinsic mechanisms of stem cells, which differ in
cell lineage, quantity, and quality, can be another source of variation.54,59,107 The same cell line has been
differentiated into the desired brain region to improve uniformity with the help of external inductive factors, but
considerable variability remained.54,58 One hypothesis is that this heterogeneity can be linked to the use of
Matrigel, an embedding material that provides supportive ECM and growth factors; Matrigel is a poorly defined
natural proteinaceous mixture that varies greatly from batch to batch.109 Compared to organoids cultured in the
Matrigel, human brain organoids produced in the brain tissue‐derived ECM hydrogel exhibited gene expression
profiles that are indicative of enhanced neurogenesis.110 Furthermore, the cortical layer development, volumetric
augmentation, and electrophysiological function of human brain organoids were further improved in a reproducible
manner, compared to matrigel organoids.
The most challenging part of current NOs is that they are not fully mature in vitro, therefore limiting the
thorough investigation of their synaptic‐integration and electrical properties.111 To date, several approaches have
been developed for the formation of synapses and functional neurons in NOs.61,112 Despite the formation of
synapses and action potential, there was insufficient robust evidence for the formation of functioning neural
networks with anatomically correct circuitry in organoids.111 The primary reasons for this could be the
inappropriate organization in organoids relative to anatomical locations in vivo, the absence of relevant cell types,
and the lack of vascular structure for oxygen and nutrient supply. A precise form of neurovascular communication
during CNS development has been found to play a critical role in the proper formation and function of the
LI ET AL. | 37
CNS.113,114 Furthermore, a series of studies highlighted the use of blood vessels as scaffolds for neuronal migration,
allowing neuronal navigation in both physiological and pathological conditions.115
4 | C H A R A C T E R I S T I C S O F NE U R O V A S C U L A R D E V E L O P M E N T
Brain functions require tremendous energy consumption and therefore a highly efficient vascular network for an
adequate supply of oxygen and nutrients.116 Studies have shown that the early stage of vertebrate CNS
development is vascularized via angiogenic ingression and the sprouting of vessel networks outside the CNS.117
Subsequently, blood vessel expansion and remodeling occur simultaneously as different neural cell types are
generated and neural circuits are established.113,117,118 Here, we focus on cortical and retinal angiogenesis during
embryonic and postnatal development.
4.1 | Development of cortical vasculature
Cortical blood vessels are derived from vessels that invade along the basal side of the cortex. Extensive branching
and capillary sprouting at various depths subsequently occur, yielding a highly complex hierarchical
pattern.116,119–121 In the mouse brain, vasculogenesis occurs simultaneously with nervous system development.
At around embryonic days 7.5–8.5 (E7.5–E8.5), the neural plate forms the neural tube.122 Subsequently, at E9.5,
blood vessels begin to sprout and invade the neuroepithelium from the perineural vascular plexus (PNVP) and
extend toward the ventricle area, where angiogenic growth factors such as vascular endothelial growth factor
(VEGF) are highly expressed.120,123 The vessels continue to give off new branches, invading the remaining areas
under embryonic development in all CNS tissues except the retina, and forming a rich capillary plexus. These
processes are concomitant with NPC proliferation and differentiation, as well as the migration of differentiated
neurons and glia (Figure 2).113,122 Briefly, neural stem cells (NSCs) give rise to NPCs that differentiate into migrating
neuroblasts, which generate immature neurons and then eventually mature neurons, oligodendrocyte precursors
(OLPs), oligodendrocytes (OLs), and astrocytes.113
Studies have identified a great quantity of signaling molecules that modulate CNS angiogenesis and blood
vessel patterning, such as NPC‐derived Wnt7a/b,124–126 Norrin (Ndp),127 VEGF,128,129 radial‐glia derived TGF‐β1,130
Vegfab/Vegfr2,131 neuron‐derived Nogo‐A,132 OLP‐derived Wnt7a/b,133 and astrocyte‐derived VEGF.134 It is worth
noting that the vasculature also contributes to the migration of γ‐aminobutyric acid (GABA)ergic neurons and
Cajal‐Retzius cells by secreting inhibitory neurotransmitters and stromal‐cell derived factor‐1 (SDF‐1),
respectively.135–137 Moreover, extracellular glycoprotein, Reelin, secreted by Cajal‐Retzius cells, plays an
indispensable role in promoting neuronal migration and lamination of the neocortex.136,138 Previous reviews have
further illustrated the mechanism by which neurons communicate with blood vessels and instruct blood vessel
formation following the requirements of human brain development.113,115 Later in development, the formation of
neurovascular units (pericytic and astrocytic endfeet surround ECs) builds up the BBB, which seals the CNS from
blood and controls the influx and efflux of a wide variety of biological substances.114,139,140
4.2 | Development of retinal vasculature
Two types of arteries supply the retina with blood: the central retinal arteries, which supply the inner retina; and the
posterior ciliary arteries, which supply the RPE and outer retina (photoreceptors). During embryogenesis, the
development of the human retinal vasculature is highly coordinated to meet the nutritional and oxygen
requirements of the developing retina.141 In humans, three‐component vasculature (hyaloid artery, vasa hyaloid
38 | LI ET AL.
F I G U R E 2 Neocortical vascularization processes. Schematic diagram indicating the increasing complexity of the
human brain over time. During neurogenesis, NSECs give rise to neuroblasts and eventually differentiate into
neurons. As neurogenesis starts, the PNVP forms. Subsequently, blood vessels begin to sprout from the PNVP and
extend toward the ventricular area. CP, cortical plate; iSVZ, inner subventricular zone; IZ, intermediate zone; MZ,
marginal zone; NSECs, neuroepithelial stem cells; oRG, outer (SVZ) radial glia; oSVZ, outer subventricular zone; SP,
subplate; tRG, truncated radial glia; vRG, ventricular (VZ) radial glia; VZ, ventricular zone. [Color figure can be
viewed at wileyonlinelibrary.com]
propria, and tunica vasculosa lentis) lies in the vitreous, initially develops around 4–6 weeks gestation (WG).141 The
hyaloid vessels, which originate from the central hyaloid artery that runs along the optic nerve, pass through the
lamina cribrosa, and enter the optic disk nasal to the postocular center.142 When the transient fetal vasculature of
the vitreous begins to regress around 13 WG, the retinal vasculature formats contemporaneously.143 Unlike in mice,
whose retinal vasculature begins to form in the first few weeks of postnatal development (Figure 3A), human retinal
vasculature development is completed before birth (38–40 WG).54,142
The retinal vessels branch into three capillary layers (superficial, intermediate, and deep). The superficial
vasculature is the first to be formed and emerged from the optic nerve head, progressing radially toward the retinal
periphery, and reaching the nasal side of the ora serrata by the eighth month of gestation (Figure 3A).144
Subsequently, retinal vessels sprout into the depth of the retina to form the deep retinal vascular layer at the base
of the outer plexiform layer (OPL). Two distinct capillary beds are formed: the deep capillary plexus in the inner
nuclear layer (INL), and the superficial capillary plexus in the ganglion cell layer (GCL). Then, the intermediate layer is
completed between the superficial and deep layers, thus forming three‐vascular patterns in these retinal vascular
plexuses (Figure 3C). Interestingly, no blood vessels from the central retinal arteries can extend into the OPL. The
photoreceptor layer of the retina and RPE are therefore free of blood vessels nourished by the central retinal artery
LI ET AL. | 39
F I G U R E 3 Schematic representation of retina vascularization processes. (A) Retinal‐vascular development from

E20 to postnatal Day 15 (P15). At E20, vessel sprouts emerge from the optic nerve head and radially extend to the
retinal periphery. The growth of the superficial vascular plexus of the retinal vasculature (red) follows the astrocytic
template (green). (B) Cross‐sectional image of an eye showing the retinal and choroid vessels. (C) An enlarged cross‐
sectional view from (B). Three retinal capillaries (the superficial retinal vessel lies in the NFL; the intermediate and
deep vascular networks lie in the IPL and OPL, respectively) are embedded among retinal neurons and the choroidal
blood vessels are located beneath Bruch's membrane. All these vessels provide oxygen and nutrients to the outer
retina. (D) The function of endothelial tip‐stalk machinery in sprouting angiogenesis. Sensitivity of the tip cell to
VEGF. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; NFL, nerve fiber layer; ONL,
outer nuclear layer; OPL, outer plexiform layer; RPE, retinal pigment epithelium; VEGF, vascular endothelial growth
factor. [Color figure can be viewed at wileyonlinelibrary.com]
and are instead supplied by the choriocapillaris from the choroid. The choriocapillaris is a high‐flow capillary
network that provides oxygen and nutrients to the outer retina. RPE and photoreceptors have high rates of
metabolism and therefore very high oxygen demand.145 The outer blood‐retinal barrier (oBRB) is formed by
choroidal capillaries in collaboration with RPE. Similar to BBB, the oBRB plays a vital role in maintaining retinal
homeostasis.146 The inner BRB (iBRB) is formed by retinal capillary ECs of the inner retina with complex tight
junctions, which can efficiently supply nutrients to the retina and remove endobiotic and xenobiotic from the retina
to maintain homeostasis in the neural retina.146–148
Human retinal vasculature development occurs via two distinct cellular processes, vasculogenesis and
angiogenesis. Vasculogenesis is defined as de novo formation of initial vascular networks.149 Angiogenesis
describes the formation of new vessels, which sprout from pre‐existing blood vessels and extend the vascular
network. Sprouting angiogenesis can take place via the endothelial tip‐stalk machinery, including stimulation of ECs
by growth factors (e.g., VEGF), migration and proliferation of ECs, and the formation of the capillary tube.142,150
Endothelial tip cells are the leading cells at the tip of vascular sprouts, guiding vessels to sprout along growth factor
gradients such as VEGF gradients (Figure 3D). In contrast, neighboring stalk cells proliferate during sprout extension
and form nascent vascular lumens, sustaining the extension and perfusion of vascular sprouting.142 Tip cells are
more VEGF‐sensitive than stalk cells, therefore holding the tip cells at the leading edge of the growing vascular
40 | LI ET AL.
plexus (Figure 1).142 Furthermore, the TGF‐β superfamily and Norrin/Wnt signaling pathways both play critical roles
in the modulation and interaction of the tip‐stalk state and deep retinal vascular development.142
It is generally accepted that retinal neurons play crucial roles in instigating, promoting, and steering
angiogenesis.142,151 Tip cells in the retina are enriched in receptors for both angiogenic (VEGF) and antiangiogenic
growth factors, formatting the neuronal guidance cue.54,152 Retinal astrocytes, spreading from the optic nerve to
the periphery, creating a template for the developing retinal vasculature.142,153 VEGF was expressed by retinal
astrocytes; however, the mediation of angiogenesis by astrocyte‐secreted VEGF is still under debate.134,154 Cell‐
specific depletion of VEGF in astrocytes does not affect developmental retinal vascularization. One possible
explanation is that VEGF is also produced by Müller cells and other neurons.153 Compared with astrocytes,
Müller cells, located in the INL, participate in the development of the deep vascular layers rather than of the
superficial vascular network.154 Moreover, Müller cells also produce pigment epithelium‐derived factor (PEDF),
which has been reported to inhibit angiogenesis.155–157 Furthermore, disruption of the VEGF signaling pathway
in amacrine and horizontal cell interneurons can lead to the formation of an unusually dense and highly
convoluted intermediate plexus.158 RGCs were found to not only regulate angiogenic growth factors for the
development of normal retinal vasculature, but also produce antiangiogenic growth factors to prevent the
misdirection of the vessel sprouting, limiting the overgrowth of the primary plexus to the nerve fiber
layer.142,159–162 Photoreceptors and RPE also negatively regulate angiogenesis and prevent the overgrowth of
vessels beyond the OPL, keeping the photoreceptor layer completely avascular.163,164 In addition,
photoreceptors produce inflammatory factors that have been found to regulate retinal vascular
development.163,165,166 Microglia has also been considered to be an essential population in the formation
and pathophysiology of the retinal vascular system.167–169 Taken together, these evidence indicate that
neuronal cells are critical for retinal vascularization.
4.3 | The role of CNS vascularization in neurogenesis and neuronal maturation
Not only do neurons exert effects on vasculogenesis and angiogenesis, but the developing vessels in turn also
influence neurodevelopmental processes by regulating neuronal genesis, migration, and differentiation.115,170,171
However, very little is known about the effects of angiogenesis on neurogenesis during CNS development.
Angiogenesis in the developing brain accompanies cerebral neurogenesis, which requires adequate delivery of
oxygen and nutrients to localized areas of active neurons to guarantee that metabolic are met.171,172 Interestingly,
NSCs reside physiologically in a hypoxic niche, which is required to maintain NSC selfrenewal and multi-
potency.173,174 The vasculature is essential not only for the delivery of nutrients and oxygen but also for the
establishment of an optimal microenvironment for NPCs. Blood vessels can anchor NPCs due to the integration of
integrin‐β1 and basement membrane laminin which are enriched on the coverage of vessels, leading to NPC division
and neocortical interneuron neurogenesis.163 Moreover, vascular‐derived molecular factors such as C‐X‐C motif
chemokine 12 (CXCL12), laminin, Jagged1, VEGF, stromal cell‐derived factor 1 (SDF1), and Wnt3a, are involved in
regulating NSC behavior, including neurogenesis and neural migration, selfrenewal, proliferation, and differentia-
tion.175–180 These evidence point to the critical role of vascular guidance in neurogenesis during CNS development.
Cakir et al. first compared neurons in vascularized human COs versus nonvascularized human COs and found that
organoid vascularization promoted neuronal maturation.181
During early embryonic retinal development, oxygen provided by the developing retinal vasculature is
considered to be a likely mediator of retinal neuron‐vessel and astrocyte‐vessel interactions during human retinal
development.182 EC‐derived platelet‐derived growth factor receptor beta (PDGFB) can activate PDGF receptor
alpha (PDGFRA) in retinal astrocytes and thereby promote proliferation and differentiation of these
astrocytes.182,183 Moreover, PDGFB also stimulates developing pericytes by targeting PDGFRB.183
LI ET AL. | 41
5 | N E X T G E N E R A T I O N OF NE U R A L O R G A N O I D S : T O W A R D
VASCULARIZA TION
Vascularization is particularly essential for oxygen preparation, nutrient supply, and efficient neural progenitor
differentiation during CNS development.28,184 Given the advantages and limitations of current NOs, vNOs could
further enhance the overall utility of organoids in research and therapies.185 Herein, we outline the advantages and
pitfalls of the various advanced approaches to generate vascularized organoids in vitro and discuss feasible future
strategies (Figure 4).
F I G U R E 4 Multiple systems for NO vascularization. (A) Generation of vNOs by codifferentiation of hESCs

during NO formation. VEGF and basic FGF (bFGF) are treated during EB formation and neural induction. After
embedding of neuroectodermal spheroids in Matrigel, cerebral organoids are fed with a neural induction media in
the presence of VEGF and Wnt7a to vascularization. (B) Generation of vNOs via coculture with ECs. Assembly of
neural and vascular aggregates or coculturing of hiPSCs/hESCs and ECs, leading to neurovascular assembloids and
vNOs, respectively. (C) Generation of vNOs on a microfluidic chip. Developing prevascularized NOs are placed on a
vascular bed (engineered ECM) embedded with ECs, which can generate a vascular structure, leading to vNOs by
vessel sprouting. (D) Generation of vNOs by 3D bioprinting. Fabricated polymerized tubular networks are
embedded in a cell containing hydrogel. The perfusable tube can be seeded with ECs to generate blood vessels.
(E) Generation of vNOs by implantation in vascularized animal tissue. The host vasculature infiltrates the graft,
leading to NO vascularization, and yielding functional vascular networks with blood flow between graft and
host. EB, embryoid body; hESCs, human embryonic stem cells; hiPSCs, human induced pluripotent stem cells;
NO, neural organoids; VEGF, vascular endothelial growth factor; vNOs, vascularized NOs. [Color figure can be
viewed at wileyonlinelibrary.com]
42 | LI ET AL.
5.1 | Genetic engineering
Recently, hESCs were engineered to ectopically express human ETS variant 2 (ETV2) and then mixed with normal
iPSCs.181 ETV2 is an essential transcription factor that is sufficient for reprogramming primary human adult skin
fibroblasts into functional ECs during neurogenesis.186 Cakir's group was the first to use this strategy to establish a
vascularized organoid system. The EB mixture generated human COs containing ECs, which in turn formed a
complex vascular‐like network in COs. The vasculature‐like structures promoted the functional maturation of
organoids as illustrated by the neuronal activity, higher action potentials were observed in vascularized COs
compared to avascular ones. Moreover, the vascularized COs acquired several BBB characteristics, including an
increase in the expression of tight junctions, nutrient transporters, and trans‐endothelial electrical resistance.
However, these methods are still suffering limitations, including a low degree of vascularization and insufficiency of
vascular cell types, such as smooth muscle cells and mesenchymal stem cells.187 Ectopic overexpression of
endothelial‐related tight junctions have been used to generate ECs from cell reprogramming.188,189 Studies from
Margariti's group showed that transferring four reprogramming factors (Oct4, Sox2, Klf4, and cellular
myelocytomatosis oncogene [c‐Myc]) into human fibroblasts for 4 days generated partial iPSCs which could
differentiate into both endothelial‐ and smooth‐muscle like cells.190,191 Notably, the overexpression of selected
endothelial‐tight junctions could be used to induce mesodermal progenitors, which further differentiated toward
endothelial or smooth‐muscle lineages under different stimulatory conditions.188
Moreover, recent work demonstrated that a combination of the overexpression of reprogramming factors and
directed differentiation can build cross‐germ‐layer and cross‐lineage organoids.187 Neuro‐vascular organoids were
generated by overexpression of NEUROD1. The neuro‐vascular organoids maintained neural and vascular function
for at least 45 days in culture. Others showed that overexpression of ATF5 and PROX1, combined with targeted
CRISPR‐based transcriptional activation of endogenous CYP3A4 advanced the maturity and vascularity of hiPSC‐
derived fetal liver organoids; however, there is no current evidence this system can be translated to NOs.192
Prevascularized NOs showed the capability of the vascular networks to connect to the host vasculature and form
perfusable blood vessels when implanted into mice brain. While genetic engineering has emerged as a promising
strategy to generate prevascularized NOs, the results are not always predictable or controllable.
5.2 | Multidirectional differentiation
During early embryonic development, the vascular and CNS systems, although generated by two different germ
layers (mesoderm and ectoderm, respectively), develop simultaneously.193 Mesoderm is the exclusive source of ECs
during embryogenesis. Previous studies have created a multistep protocol for modulating mesodermal development
and vascular specifications. The protocol starts with the induction of glycogen synthase kinase 3 (GSK3) inhibitor
CHIR99021, which activates the Wnt signaling pathway; next, organoids are treated with VEGF, BMP4, or FGF2 to
induce vascular progenitor differentiation.189,194 In 2019,Penninger et al. optimized the protocol for the
differentiation of hPSCs into vascular organoids containing ECs and pericytes, which selfassembled into capillary
networks.155 Transplantation of these organoids into mice allowed the formation of a stable and perfused vascular
tree (arteries, arterioles, and venules). Moreover, chamber‐like structures, cardioids, were generated from hPSCs
and revealed selforganizing principles of human cardiogenesis.195 Therefore, promoting the codifferentiation of
stem cells into mesoderm and ectoderm is another possible strategy to prevascularize NOs.
Recent studies have modified the protocol to elongate hPSC aggregates, which preferentially differentiate into
neuroepithelium with the negligible presence of mesodermal and endodermal phenotypes.43 Ham et al. have
demonstrated that exogenous VEGF treatment can be used to induce codifferentiation of ECs without inhibiting
neural morphogenesis, leading to the formation of blood vessel‐like structures in cerebral organoids (Figure 4A).196
The results also showed that tube formation, vasculogenesis, and mature BBB characteristics of cerebral organoids
LI ET AL. | 43
were regulated by VEGF.196 However, limited expansion of blood vessels was found in cerebral organoids as the
sparse vascular‐like structures in long‐term cultures. Using an analogous co‐differentiation approach, Holloway
et al. modified culture conditions, resulting in a population of ECs that presented early in hiPSC‐derived intestinal
organoids despite endodermal induction.197 VEGF, BMP4, and FGF2 were applied to modify media conditions to
expand and maintain endogenous ECs within organoids, keeping the highest transcriptional similarity to native
intestinal ECs.197 Taken together, these data demonstrate that modified media can be utilized to create vessel‐like
structures in cerebral organoids in vitro.
Multidirectional differentiation of stem cells shows great promise for organoid vascularization due to
mesoderm and ectoderm sharing the same stem cell origin. This methodology, however, is hampered by the limited
knowledge of CNS differentiation protocols, as well as by mesodermal differentiation. The timing of mesoderm
introduction and EC differentiation should ideally mimic the development of nascent vasculature during embryonic
neurogenesis in vivo. Moreover, the percentages of ECs and tissue‐specific ECs, location of ECs, and even the
cytoarchitectural organization of brain organoids are hard to control.198 Although a population of ECs were
successfully induced within organoids early during codifferentiation, these cells are not well maintained during
prolonged culture under standard growth conditions.197 This approach could be less efficient than mixing in ECs
directly or when overexpression of ETV2 is used.199 Therefore, modified culture conditions are indispensable for
supporting the survival of this population of ECs within NOs for long‐term culture.
5.3 | Coculture with endothelial cells
Takebe's lab reported one of the first instances of organoid vascularization. They generated vascularized liver buds
by combining human umbilical vein endothelial cells (HUVECs), human bone marrow mesenchymal stem cells
(MSCs), and specific parenchymal‐cell types.200 Campisi et al. designed a coculture complex system with a triculture
of iPSC‐derived ECs, astrocytes, and pericytes.201 In this protocol, a perfused vasculature in a microfluidic device
was used to form a selforganized microvascular model of the BBB. Subsequently, iPSC‐derived brain organoid was
re‐embedded in Matrigel with ECs, which derived from the same iPSCs.202 Pham's group found that the integration
of brain organoids with ECs resulted in robust vascularization of the organoids after 3–5 weeks of in vitro and
2 weeks of in vivo culture.202 Human CD31‐positive vascularized structure was found inside the organoid after
transplantation (Figure 1). However, there is no evidence showing the functional connectivity of the organoid
capillaries with the host brain or perfusion of the human capillaries with rodent blood.202
In 2020, Shi et al. generated vascularized human COs by coculturing HUVECs with hESCs or hiPSCs in vitro
(Figure 4B).2,31 A well‐developed mesh‐like or tube‐like structure was formed in the COs. The vascularized COs
recapitulated human neocortical development, presenting similar cell types (RGs, oRGs, induced pluripotent cells
[IPC], excitatory neurons, interneurons, astrocytes, and microglia) in vivo. Moreover, organoids can survive in the
culture system over 200 days and generate spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs
and sIPSCs) as well as bidirectional electrical transmission, suggesting the presence of chemical and electrical
synapses within the organoids. Although human primary cell lines can be used to vascularize organoids, it is
desirable to vascularize organoids from the same hPSCs.203,204 Recently, Worsdorfer et al. described a protocol to
incorporate stromal components by coculturing them with iPSC‐derived mesodermal progenitor cells, leading to
neuro‐mesenchymal organoid assembly and vNOs maturation (Figures 1 and 4B).205 However, a certain variability
was observed in the density and pattern of the vascular network and the extent of endothelial sprouting into NOs.
This approach, however, raises several questions about the initial ECs ratio in each brain organoid, including the
EC types (endothelial progenitors or tissue‐specific ECs) and the timing of coculture, as well as the realistic time
point when ECs take part in the in vivo embryonic neurogenesis (should ECs be added once the aggregates are
established?).198 Also, ECs alone are insufficient to recreate the full vascular structure. In addition, the successful
formation of vascular networks from ECs still requires the coculture of VEGF‐secreting cells or exogenous VEGF to
44 | LI ET AL.
activate sprouting angiogenesis via endothelial tip‐stalk machinery. Moreover, a direct mixing of stem cells and ECs
may fail to form proper EBs or lead to uncontrollable differentiation due to the cross‐talk between stem cells and
ECs. In this case, the interaction between stem cells and ECs needs to be considered.
5.4 | Microfluidics and 3D bioprinting
Researchers have begun integrating bioengineering approaches with CNS organoid derivation protocols to produce
vascularized organoids more effectively. As reviewed previously, numerous engineering methodologies can be
applied to cerebral organoid culture to generate vasculature.27,206,207 3D human lung fibroblast spheroid with a
perfusable vascular network in a microfluidic device was generated in Nashimoto's lab by mixing HUVECs with
spheroids.208 In this microchannel, HUVECs on both side walls invaded into the gel as angiogenic sprouts, reaching
the vessel‐like structures in a spheroid to form a continuous lumen. Homan et al. recently developed a millifluidic
device engineering with the supportive ECM to facilitate the emergence of vascular cells in the organoids, as well as
the emergence of kidney‐like morphology.209 Developing kidney organoids were placed on ECM, housed within a
perfusable microfluidic chip, and subjected to controlled fluidic shear stress (Figure 4C). Hemodynamic shear stress,
the frictional force acting on vascular ECs, was critical for the maintenance of the molecular signature and
phenotype of ECs.210,211 The shear stresses exerted by flow greatly expanded the endogenous pool of endothelial
progenitor cells and generated vascular networks with perfusable lumens surrounded by mural cells.209 Compared
with control organoids in a static, no‐flow condition, vascularized kidney organoids under flow showed more
mature podocyte and tubular compartments. However, fluidic shear stress has yet to be applied to recapitulate the
mechanical environment that favors ECs to create vNOs in vitro.
Recently, Yue et al. successfully reconstituted the BBB model by applying a microfluidic perfusion system and a
3D interconnected network.212 After reseeding ECs inside the channels of the network, the network was wrapped
by collagen matrix combined with coculturing with pericytes, astrocytes, and neurons. From this system,
researchers derived vascularized neural constructs that successfully reconstituted BBB function. Miller et al. printed
mold cylindrical networks that could be lined with endothelial tip‐stalk and perfused with blood under high‐pressure
pulsatile flow.213 The molds could be dissolved and endothelialized to create large‐scale vascularized tissues with
tunable vascular geometries (Figure 4D). To date, numerous biocompatible materials combined with 3D bioprinters
have been used to create a temporary sacrificial scaffold. Therefore, novel photosensitive, PH‐ and temperature‐
sensitive biodegradable polymers are increasingly appreciated. However, the limitations of the current sacrificial
template are the challenges of dimensional accuracy and precision, in particular for capillary‐sized vessels.27
5.5 | In vivo organoid transplantation
Although following the strategies presented above might enable vessel‐like tubular networks to be formed within
NOs and other neural structures, the issues of oxygen/nutrient supply and authentic blood microenvironment need
to be solved.28,185 Therefore, an alternative approach might be engrafting the organoids into an animal host, leading
to the ingression of vessels into the grafting organoids and the formation of perfusable vascular structures. In a first
demonstration, vascularized and functional human liver was generated from hiPSCs by transplantation of 3D liver
buds.214 Subsequently, Mansour et al. provided a method for transplanting human 40‐ to 50‐day‐old brain
organoids into the retrosplenial cortices of ~5‐week‐old NOD‐SCID (nonobese diabetic/severe combined
immunodeficient) mice (Figure 4E).28 The host blood vessels were observed to invade the organoid grafts, leading
to progressive neuronal temporal differentiation and maturation, axonal growth, and gliogenesis in the host brain.
Interestingly, electrophysiological activity was detected in organoid grafts, and functional synaptic connectivity of
graft‐to‐host was established between the vascularized grafts and host brain tissues.28 In another study, 60‐day‐old
LI ET AL. | 45
human prevascularized cortical organoids cocultured with HUVECs were intracerebrally implanted into the S1
cortices of immunodeficient mice and could yield functional vascular networks with blood flow between graft and
host.31 Moreover, in vivo transplantation of prevascularized CNS organoids‐derived coculturing also could generate
more robust vascularization of the organoids after transplantation in vivo.181,202
A major advantage of organoid transplantation in vivo is that the host brain provides an appropriate
microenvironment, such as vascular circulation. However, a prerequisite for the successful application of this
approach is sufficient prevascularized NOs. Even though rapid advances in transplantation, this approach could be
hindered by limited knowledge of transplant outcomes of prevascularized NOs on human beings in current
literature. Furthermore, the application of this method is restricted in whole‐RO transplantation due to the retinal
vascular development and anatomy are precisely controlled.
6 | C O N C L U D I N G R E M A R K S AN D F U T U R E P E R S P E C T I V E S
NOs hold tremendous promise for addressing the development, physiology, and diseases of human CNS, as well as
application in translational medicine. However, organoids currently still suffer from variability in cytoarchitectural
organization, cellular phenotypes, and physiological maturation. Therefore, differences between organoids and
primary cell types could give rise to misleading assumptions about neuropathology and misinform therapeutics.215
Moreover, NO‐derived cells applied in transplantation therapy need further consideration.
Indeed, the incorporation of ECs, mesenchymal cells, and inflammatory cells into NOs improves their
maturation and might be required to create fully functional microtissues that accurately recapitulate
neurodevelopmental conditions in vivo. To overcome the biggest obstacle, of serious necrosis occurring within
NOs due to insufficient oxygen and nutrient perfusion, important advances have been made in the last few years to
enhance the development of next‐generation organoid vascularization. It is worth mentioning that although
coculture with iPSC‐derived ECs or tissue‐specific ECs, codifferentiation of stem cells, gene engineering or
bioprinting, and in vivo organoid transplantation are indeed breakthrough methodologies in organoid vasculariza-
tion, these methods cannot generate complete vascular systems. None of the formed EC networks within organoids
have been perfused throughout in vitro, and these vascular networks still lack blood vessel functionality. Moreover,
the vascularized networks generated in organoids are irregular and disarranged, rendering them unable to mimic the
stratified distribution and the direction of blood vessels in vivo. In particular, a well‐organized network with three
retinal vascular layers in the retina was established (Figure 3), but, no blood vessels from the central retinal arteries
could extend into the OPL. Substantial efforts are also needed to unveil the mysterious vascular pattern in brain
tissues. Therefore, optimal approaches should be based on developmental principles of developing, anatomical, and
site‐specific vasculature when generating vNOs.27 Angiogenesis in brain tissues involves not only ECs but also
other cell types such as pericytes, vascular smooth‐muscle cells, and immune cells. Astrocytes in particular, which
provide a critical structural template for vessel sprouting, should also be considered.
Given the results of previous studies, we believe that the integration of superior technologies, including gene
engineering, multidifferentiation, coculture systems, microfluidics, and 3D bioprinting as well as approaches that
combine the strengths of these varying methods can ultimately generate mature and functional vNOs. Considering
vascularization facilitates the delivery of oxygen and nutrients, the metabolically activities/networks such as
mitochondrial respiration, glucose, fatty acid, and amino acid metabolism in vNOs will offer invaluable information
that is difficult to acquire in previous iPSC‐derived disease models.216,217 Multiomic technologies such as genomics,
transcriptomics, proteomics, and metabolomics can help to unravel the developmental events mediated by
neurovascular interactions in vNOs. Given the interaction between angiogenesis and neurogenesis, vasculature
developmental principles must be considered for organoid vascularization. NOs generated under such
circumstances can serve as ever‐more‐reliable models in vitro for translational and regenerative applications.
46 | LI ET AL.
A C KN O W L E D G M E N T S
This study was supported by the National Key Research and Development Program of China grants
2018YFA0107302; the National Natural Science Foundation of China grants 81873688 and 31930068.
CO NFL I CT OF INTERES T
The authors declare no conflict of interest.
D A TA A V A I L A B I L I T Y S T A T E M E N T
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.
ORCID
Haiwei Xu http://orcid.org/0000-0002-8840-7918
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A U T H O R B I O G R A P H IE S
Minghui Li obtained his Master's degree from Shantou University. Subsequently, he studied in the Department
of Pathology & Medical Biology at the University Medical Center Groningen (UMCG, The Netherlands). He
joined the Visual Damage and Regeneration group in Southwest Hospital, Third Military Medical University
(Army Medical University) and Key Lab of Visual Damage and Regeneration and Restoration of Chongqing,
Chongqing, China. He is now focusing on stem cell‐derived retinal organoids and organoid vascularization.
Lixiong Gao received his PhD degree at the Army Medical University in 2017. He currently works as an
ophthalmologist in the 3rd Medical Center of PLA General Hospital. His researches mainly focus on the
mechanism of retinal degenerative diseases and stem cell‐based cell therapy.
Ling Zhao obtained her PhD from Peking University. She received her Postdoctoral training in Shiley Eye Center
at the University of California, San Diego from 2010 to 2014. Currently, she is a Principle Investigator in the
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat‐sen University. She uses
genetics to guide mechanism studies and design therapies for human vision diseases, especially for age‐related
blindness, and provides novel insights into the underlying genetic mechanisms in the pathogenesis of common
54 | LI ET AL.
complex eye diseases. Also, she has contributed to epigenetic studies on quantitative prediction of human aging
rate and age‐related blindness.
Ting Zou received her PhD degree from the Third Military Medical University in 2019. She became an associate
research fellow in Southwest Hospital/Southwest Eye Hospital (2020), Third Military Medical University (Army
Medical University) and Key Lab of Visual Damage and Regeneration and Restoration of Chongqing, Chongqing,
China. Currently, she is mainly engaged in basic research and clinical translational research of stem cell therapy
for retinal degenerative diseases, and deeply explores the interaction between transplanted stem cells and the
retinal degenerative microenvironment.
Haiwei Xu received his PhD degree from the Third Military Medical University in 2001. Then he studied at the
Center for Nuclear Receptors and Cell Signaling, the University of Houston as visiting scholar. In 2011, he
joined the Visual Damage and Regeneration group in Southwest Hospital/Southwest Eye Hospital, Third
Military Medical University (Army Medical University) and Key Lab of Visual Damage and Regeneration and
Restoration of Chongqing as Principle Investigator and Vice Director. His current research interests include
basic and clinical translational research of stem cell therapy for retinal degeneration diseases, conducting in‐
depth exploration of stem cell therapy for retinal degeneration diseases and injury mechanisms, and
nanoparticle‐eye toxicity research.
S UP P O R T I N G I N F O R M A T I O N
Additional supporting information can be found online in the Supporting Information section at the end of this
article.
How to cite this article: Li M, Gao L, Zhao L, Zou T, Xu H. Toward the next generation of vascularized human
neural organoids. Med Res Rev. 2023;43:31‐54. doi:10.1002/med.21922

Medicinal Research Reviews - 2022 - Li - Toward The Next Generation of Vascularized Human Neural Organoids

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Medicinal Research Reviews - 2022 - Li - Toward The Next Generation of Vascularized Human Neural Organoids

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Received: 20 October 2021 | Revised: 22 February 2022 | Accepted: 9 August 2022

Toward the next generation of vascularized

Minghui Li1,2 | Lixiong Gao3 | Ling Zhao4 | Ting Zou1,2 |

Med Res Rev. 2023;43:31–54. wileyonlinelibrary.com/journal/med © 2022 Wiley Periodicals LLC. | 31

will summarize recent technological advances in culture

2.1 | Generation and characterization of cortical organoids

2.2 | Generation and characterization of retinal organoids

3 | CURRENT CHALLENGES OF HUMAN NEURAL

4.1 | Development of cortical vasculature

4.2 | Development of retinal vasculature

F I G U R E 3 Schematic representation of retina vascularization processes. (A) Retinal‐vascular development from

4.3 | The role of CNS vascularization in neurogenesis and neuronal maturation

F I G U R E 4 Multiple systems for NO vascularization. (A) Generation of vNOs by codifferentiation of hESCs

5.1 | Genetic engineering

5.2 | Multidirectional differentiation

5.3 | Coculture with endothelial cells

5.4 | Microfluidics and 3D bioprinting

5.5 | In vivo organoid transplantation

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